ATP6AP2 is robustly expressed in pancreatic β cells and neuroendocrine tumors, and plays a role in maintaining cellular viability

ATP6AP2, also known as (pro)renin receptor, has been shown to be expressed in several tissues including pancreatic β cells. Whereas ATP6AP2 plays an important role in regulating insulin secretion in mouse pancreatic β cells, the expression profiles and roles of ATP6AP2 in human pancreatic endocrine cells and neuroendocrine tumor cells remain unclear. Here in this study, we investigated the expression profiles of ATP6AP2 in pancreatic endocrine cells, and found that ATP6AP2 is robustly expressed in pancreatic insulinoma cells as well as in normal β cells. Although ATP6AP2 was also expressed in low-grade neuroendocrine tumors, it was not or faintly detected in intermediate- and high-grade neuroendocrine tumors. Knockdown experiments of the Atp6ap2 gene in rat insulinoma-derived INS-1 cells demonstrated decreased cell viability accompanied by a significant increase in apoptotic cells. Taken together, these findings suggest that ATP6AP2 plays a role in maintaining cellular homeostasis in insulinoma cells, which could lead to possible therapeutic approaches for endocrine tumors.

www.nature.com/scientificreports/ and alternative treatments are medications, such as somatostatin analogues, streptozotocin and everolimus [15][16][17] , although no medication can fully inhibit tumor progression and prevent subsequent hypoglycemia. As the suppression of ATP6AP2 has been reported to induce apoptosis in various types of tumors 5,6 , as described above, we hypothesized that a loss-of-function approach for Atp6ap2 may also inhibit the tumor growth in insulinoma cells. Therefore, we analyzed the expression profiles of ATP6AP2 in pancreatic endocrine cells and insulinoma cells, and aimed to clarify whether the knockdown approach for Atp6ap2 can reduce the viability of insulinoma cells, which may lead to the development of novel therapeutic approaches.

Results
ATP6AP2 proteins are expressed in human pancreatic endocrine cells. It has been reported that V-ATPase and its accessory protein ATP6AP2 are expressed in mouse islets 11,18 . In addition, single-cell RNA sequencing of human pancreata from five healthy subjects demonstrated that ATP6AP2 mRNAs are highly expressed in various types of endocrine cells in the pancreas 19 . To further confirm that the ATP6AP2 protein is indeed expressed in human endocrine cells, including tumor cells, immunohistological analysis was performed in human pancreata from patients with insulinoma and nonfunctioning NET. The expression of ATP6AP2 proteins was clearly detected in insulin-expressing cells of normal islets near the insulinoma lesions (Fig. 1a,b) and nonfunctioning NETs (Fig. S1), whereas it was not or faintly detected in glucagon-expressing cells and somatostatin-expressing cells in the same regions. In addition, immunostaining in rat insulinoma cell line INS-1 cells demonstrated ATP6AP2 expression in the cytosol of INS-1 cells (Fig. S2).
ATP6AP2 proteins are expressed in insulinomas and nonfunctioning NETs. To further investigate whether ATP6AP2 proteins are expressed in endocrine tumor cells in the pancreata, immunohistological analysis was performed in human pancreata from 10 patients with insulinoma and 31 patients with nonfunctioning NET. The clinical characteristics of the patients are listed in Table 1 and the Supplementary Tables. Immunostaining against ATP6AP2 demonstrated the robust expression of ATP6AP2 in insulinoma cells as well as in non-tumor islet cells of the same section, whereas non-tumor cells surrounding the insulinoma cells were not stained with ATP6AP2 ( Fig. 2a-c). Coimmunostaining against ATP6AP2 and insulin demonstrated that all ATP6AP2-expressing cells overlapped with insulin (Fig. 2d). These findings suggest that ATP6AP2 proteins are highly and specifically expressed in insulin-expressing cells in pancreatic sections of insulinoma. Furthermore, immunoblotting against ATP6AP2 demonstrated a significantly higher level of ATP6AP2 protein expression in INS-1 cells compared with in islets isolated from C57BL/6 J mice (Fig. 2e, Fig. S3).
We further performed immunostaining against ATP6AP2 in pancreatic sections from patients with nonfunctioning NET. Whereas low-grade (G1) NET cells were clearly stained for ATP6AP2 (Fig. 3a-c), G2/G3 NET cells in which Ki67 index was greater than 10%, were hardly stained for ATP6AP2 ( Fig. 3d-f, Table 2, Fig. S4). Immunostaining for ATP6AP2 and insulin in different types of NET demonstrated that ATP6AP2 proteins were clearly detected in normal islets and tumor cells in G1 NET, but not in G2/G3 NET cells (Fig. S3), which suggests that the expression level of ATP6AP2 is likely to be correlated with proliferation capacity of NET cells, and can be a therapeutic target in certain types of NETs.

Suppression of ATP6AP2 gene expression induces the apoptosis of INS-1 cells. To investigate
the role of ATP6AP2 in insulinoma cells, ATP6AP2 mRNAs were knocked down in the rat insulinoma cell line INS-1 using small interfering RNAs (siRNAs). As shown in Fig. 4a, both Atp6ap2 siRNA-1 and siRNA-2 significantly suppressed the expression of Atp6ap2 mRNAs compared with scrambled siRNAs. In addition, immunoblot analysis resulted in a significant decrease in ATP6AP2 proteins by Atp6ap2 siRNA-1 (Fig. 4b, Supplementary  Fig. 3). When cell viability of INS-1 cells was evaluated by the WST-1 assay, there was no significant difference in cell viability between cells treated with Atp6ap2 siRNA-1 and those treated with scrambled siRNAs (Fig. 4c), whereas terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining demonstrated that the number of TUNEL-positive cells was significantly increased by Atp6ap2 siRNA-1 treatment (Fig. 4d). In addition, 5-ethynyl-2-deoxyuridine (EdU) staining showed that the number of EdU-positive cells was significantly decreased in the cells treated with Atp6ap2 siRNA-1 (Fig. 4e,f). Taken together, these results suggested that ATP6AP2 likely plays a role in maintaining the cellular viability of insulinoma cells.

Suppression of Atp6ap2 gene expression and a somatostatin analogue nonadditively induced apoptosis.
To further investigate whether the suppression of Atp6ap2 gene expression shows any synergistic effects in combination with already established anti-tumor treatments, INS-1 cells were treated with Atp6ap2 siRNA-1 and/or octreotide, a somatostatin analogue, and the number of apoptotic cells was sequentially quantified with IncuCyte®, a high-throughput device that can acquire and view culture cell images over time. Whereas both treatment with Atp6ap2 siRNA-1 and octreotide significantly increased the number of apoptotic cells compared with treatment with scrambled siRNAs (Fig. 5a,b), which was confirmed by TUNEL staining (Fig. 5c), there were no additive effects of Atp6ap2 siRNA-1 and octreotide treatment on cellular apoptosis.

Discussion
While ATP6AP2 has been reported to play a role in regulating the viability of various types of tumor cells, such as pancreatic cancer 5 and glioblastoma 6 , the expression profiles of ATP6AP2, and the potential role of ATP6AP2 in pancreatic endocrine cells, including tumor cells, remains unclear. In this study, histological analysis demonstrated the robust expression of ATP6AP2 in human β cells, which is consistent with previous findings in mouse and human pancreata 11,18,20 . Whereas single-cell RNA sequencing resulted in the widespread expression of ATP6AP2 mRNAs in pancreatic endocrine cells as well as acinar cells 19  www.nature.com/scientificreports/ levels of ATP6AP2 proteins in α cells, δ cells, and acinar cells in our present study (Fig. 1). Therefore, it is possible that the post-transcriptional modification of ATP6AP2 differs between β cells and non-β-cells in the pancreas. On the other hand, another previous study reported that the ATP6AP2 protein was detected in a proportion of α cells as well as in β cells in human islets 20 , which implies that ATP6AP2 can be expressed in some α cells under certain conditions. ATP6AP2 was found to be expressed not only in pancreatic β cells but also in insulinoma cells in all patients (Fig. 2, Table 2). On the other hand, in some patients with nonfunctioning NETs, in which the Ki67 index was greater than 10%, there were few cells stained for ATP6AP2 ( Fig. 3d-f, Table 2), showing that the expression of ATP6AP2 was negatively correlated with tumor growth in nonfunctioning NETs. This is in contrast with previous studies, which showed that ATP6AP2 expression was positively correlated with the Ki67-labeling index 6 , and that systemic administration of anti-ATP6AP2 antibodies into mice bearing subcutaneous pancreatic adenocarcinoma cells significantly decreased the number of Ki67-positive cells 21 . In addition, EdU-staining demonstrated that the suppression of Atp6ap2 gene expression inhibited a proliferation of INS-1 cells (Fig. 4e), which is consistent with previous reports 6, 21 . Whereas Atp6ap2 knockdown resulted in no significant effects in the WST-1 assay www.nature.com/scientificreports/ ( Fig. 4c), it significantly increased the number of TUNEL-positive cells (Fig. 4d). As the WST-1 assay reflects metabolic activity as well as cell viability, compensative metabolic processes may affect the results of the WST-1 assay. Further metabolomics analysis would provide better insight into the molecular mechanisms underlying these results. As it has been reported that Atp6ap2 deletion does not induce cellular apoptosis in primary β cells 11 , it is possible that the role of ATP6AP2 in the cell viability may be different between primary β cells and insulinoma cells. Furthermore, the expression levels of Atp6ap2 mRNAs was shown to be positively associated with the progression of pancreatic ductal adenocarcinoma 22 . These results imply that the role of ATP6AP2 in cellular proliferation may depend on the cell types and features of tumor cells. These findings, together with in vitro data that the suppression of Atp6ap2 gene expression induces apoptosis in INS-1 insulinoma cells (Figs. 4 and 5), suggest that the Atp6ap2 gene may be a possible therapeutic target for insulinoma and early stage NETs, but not for advanced NETs with higher proliferation activity. The downregulation of ATP6AP2 resulted in increased cellular apoptosis in insulinoma cells (Fig. 5), which is consistent with previous findings in other cell types, such as pancreatic ductal adenocarcinoma (PDAC) cells 5 , bronchial epithelial cells 23 , and nephron cells 24 . Silencing of ATP6AP2 mRNAs in PDAC cells and bronchial epithelial cells attenuated activation of the Wnt/β-catenin signaling pathway, and increased apoptosis 5,23 . Further studies will be needed to clarify the possible association between ATP6AP2 and the Wnt/β-catenin signaling pathway in pancreatic β cells and NET cells.
As ATP6AP2 is highly expressed in many cell types and regulates their viability, it is possible that the suppression of ATP6AP2 will have critical effects on cell viability, similarly to cytotoxic anticancer agents, such as cisplatin, gemcitabine, irinotecan and paclitaxel, having many adverse effects. On the other hand, such cytotoxic agents have been shown to have beneficial effects on killing cancer cells, beyond their adverse effects. As tumor cells in G1 NETs express high levels of ATP6AP2, optimal levels of ATP6AP2 suppression are expected to target NET cells without having any critical adverse effects in other cell types.
Atp6ap2 siRNAs induced the cellular apoptosis in insulinoma cells as potently as the somatostatin analogue octreotide, although there were no additive effects between Atp6ap2 siRNAs and octreotide (Fig. 5). Somatostatin analogues, such as octreotide and lanreotide, can be used as the first-line therapy for malignant insulinomas due to their antiproliferative effect 25 . While somatostatin analogues (SSAs) are able to control symptoms in 35-50% of insulinoma patients, their efficacy is limited partly because of tachyphylaxis 26 . Furthermore, other medications, such as streptozotocin and everolimus, cannot completely suppress tumor growth and subsequent hypoglycemia in patients with insulinoma. Therefore, it is expected that targeting the Atp6ap2 gene would be an additional option for patients with insulinoma who cannot be cured by current therapies. Other synergistic interactions between Atp6ap2 suppression and various medications should be investigated in the future for the development of more effective strategies to cure insulinoma and pancreatic NETs.

Materials and methods
Clinical data and tissue specimens. Specimens of insulinoma and nonfunctioning NET were obtained from 41 Japanese patients, who underwent surgical resection at Kitasato University Hospital between 2006 and 2020. 10 patients with insulinoma were diagnosed based on typical clinical features during hypoglycemia and high insulin levels. A histological diagnosis of insulinoma and nonfunctioning NETs was based on hematoxylin and eosin staining, and immunostaining. The study protocol and a waiver of written informed consent were approved by Clinical Research Review Board of The Kitasato Institute (study approval no.: B20-377). Patients' data was obtained through an opt-out methodology. All study methods were performed in accordance with the relevant guidelines and regulations of this organization as well as the Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan.